Immobilized pH gradients: effect of salts, added carrier ampholytes and voltage gradients on protein patterns

Salts formed from strong acids and bases (e.g. NaCl, Na2SO4, Na2HPO4), present in a protein sample applied to an immobilized pH gradient (IPG) gel, induce protein modification (oxidation of iron moiety in hemoglobin) already at low levels (5 mM) and irreversible denaturation (precipitation) at higher levels (greater than 50 mM). This effect is due to production of strongly alkaline cationic and strongly acidic anionic boundaries formed by the splitting of the salt's ion constituents, as the protein zone is not and can not be buffered by the surrounding gel until it physically migrates into the gel matrix. Substitution of "strong" salts in the sample zone with salts formed by weak acids and bases, e.g.. Tris-acetate, Tris-glycinate, Good's buffers such as (N-[2-acetamido]-2-iminodiacetic acid (ADA), (2-[(2-amino-2-oxoethyl)-amino] ethanesulfonic acid (ACES), (3-[N-morpholino]propane sulfonic acid (MOPS), essentially abolishes both phenomena, oxidation and irreversible denaturation. Suppression of "strong" salt's effects is also achieved by adding, to the sample zone, carrier ampholytes in amounts proportional to the salt present (e.g. by maintaining a salt: carrier ampholytes molar ratio of at least 1:1). This suppression is due to the strong buffering power of the added carrier ampholytes, able to counteract drastic pH changes in the two moving boundaries. A reduction of these deleterious effects of strong salts is also achieved when the IPG run is performed at low voltage for a prolonged time (4 h at 500 V instead of only 1 h at 500 V, before switching to high-voltage settings). Guidelines are given for trouble-free IPG operations.